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Molecular Recognition in Antibody Engineering. Studies on recombinant and phage displayed antibodies

Malmborg Hager, Ann-Christin LU (1996)
Abstract (Swedish)
Popular Abstract in Swedish

Denna avhandling handlar om antikroppar. Antikroppar är proteiner som bildas av vårt immunförsvar för att skydda oss mot farliga ämnen i vår omgivning, som tex bakterier. På grund av antikroppars unika uppbyggnad och funktion utnyttjas ofta dessa på konstgjord väg. De kan injiceras i kroppen för att bota någon sjukdom eller användas på laboratoriet i olika analyser. Innan detta är genomförbart måste antikropparna emellertid tillverkas. Ett sätt att tillverka antikroppar är att spruta in det ämne antikroppar önskas emot, i en mus. Detta ämne brukar kallas antigen. Efter att musens eget immunförsvar producerat antikroppar mot det främmande ämnet, tappas musen på blod. I blodet finns antikropparna,... (More)
Popular Abstract in Swedish

Denna avhandling handlar om antikroppar. Antikroppar är proteiner som bildas av vårt immunförsvar för att skydda oss mot farliga ämnen i vår omgivning, som tex bakterier. På grund av antikroppars unika uppbyggnad och funktion utnyttjas ofta dessa på konstgjord väg. De kan injiceras i kroppen för att bota någon sjukdom eller användas på laboratoriet i olika analyser. Innan detta är genomförbart måste antikropparna emellertid tillverkas. Ett sätt att tillverka antikroppar är att spruta in det ämne antikroppar önskas emot, i en mus. Detta ämne brukar kallas antigen. Efter att musens eget immunförsvar producerat antikroppar mot det främmande ämnet, tappas musen på blod. I blodet finns antikropparna, som sedan kan renas fram. Problemet med detta förfarande är att om antikropparna ska användas till att sprutas in i en människa, så reagerar människans immunförsvar med att bilda antikroppar mot musantikropparna och stöter bort dessa innan de har hunnit göra någon nytta. Att tillverka antikroppar i människor, för att människan inte ska uppfatta dem som främmande och stöta bort dem, vore därför optimalt. Detta fungerar bra, men bara för ett begränsat antal främmande ämnen. Man kan ju själv tänka sig att ingen människa skulle vilja ha tex HIV insprutat i sig för att på så sätt få fram antikroppar. Nya, finurliga sätt att tillverka mänskliga antikroppar på måste i stället utvecklas. En framkomlig väg är att producera antikroppar i bakterier, vilket är vad denna avhandling handlar om. Artikel IV, V och VI handlar om hur vi försöker ta fram antikroppar, med förutbestämda egenskaper, ur stora konstgjorda antikroppsbibliotek. Artikel I, II och III handlar om hur dessa egenskaper kan mätas med hjälp av moderna apparater s.k. biosensorer. (Less)
Abstract
In vitro generation of antibody fragments of desired specificity and affinity plays an important role since it permits the generation of reagents which may be valuable for diagnostic and therapeutic applications. Furthermore, the kinetic parameters for an antibody-antigen interaction, rather than the affinity, has shown to correlate with biological functions like virus neutralization. This points at the importance of being able to evaluate kinetic parameters. In this thesis we have evaluated the BIAcore biosensor for measuring affinity and kinetic constants and pointed at events where precautions need to be taken. The recently launched ORIGEN Analyser was, furthermore, evaluated for measuring affinity constants and a protocol for measuring... (More)
In vitro generation of antibody fragments of desired specificity and affinity plays an important role since it permits the generation of reagents which may be valuable for diagnostic and therapeutic applications. Furthermore, the kinetic parameters for an antibody-antigen interaction, rather than the affinity, has shown to correlate with biological functions like virus neutralization. This points at the importance of being able to evaluate kinetic parameters. In this thesis we have evaluated the BIAcore biosensor for measuring affinity and kinetic constants and pointed at events where precautions need to be taken. The recently launched ORIGEN Analyser was, furthermore, evaluated for measuring affinity constants and a protocol for measuring dissociation rate constants was developed. The ability to select antibodies from phage displayed antibody libraries based on their kinetic parameters would be valuable. Two approaches in this direction were taken. The first was to use BIAcore biosensor for selection. By collecting elution fractions of an injected phage displayed antibody library, it could be shown that the time of dissociation is proportional to the dissociation rate constant. The second approach was to develop a modified protocol for SAP selection. SAP (selection and amplification of phage) links specific interaction to phage infection, by displaying the antibody fragment on a non-infectious phage and the antigen, fused to protein3, is added free in solution. Thus, only specific phages are allowed to infect and are amplified. We showed that addition of competing antigen to the interaction between antigen-protein3 fusion protein and a model phage displayed antibody library favoured low dissociation rate constants. Furthermore, a reduction in interaction time favoured high association rate constants. Thus, it was, for the first time, possible to select antibody fragments based on association rate. The last part of the thesis presents a new concept, which integrates phage and bacterial display in an attempt to physically link the genetic information of specifically interacting antibody-antigen molecules. This was performed by expressing peptide antigens on the surface of E. coli F pilus. The peptides were expressed as a fusion to pilin, the building block of pilus and encoded by the traA gene, which completely blocked protein3-mediated wild type phage infection. However, when a phage displayed scFv antibody was allowed to interact with pilus displayed peptide, we obtained a bacterial infection mediated by the specific antibody-antigen interaction. Thus, specific interaction on a protein-ligand level could be genetically rescued as a cellular linkage of the involved genes.This principle could have the potential to allow for screening of specific molecular interactions by crossing one phage and one bacterial library. (Less)
Please use this url to cite or link to this publication:
author
opponent
  • Prof. Uhlén, Mathias
organization
publishing date
type
Thesis
publication status
published
subject
keywords
transplantation, affinity, antibody, kinetic, Immunologi, serologi, serology, Immunology, Bioteknik, Biotechnology, library, selection, surface display, phage display, association rate, dissociation rate
pages
49 pages
publisher
Ann-Christin Malmborg, Dept. of Immunotechnology, P.O.Box 7031, S-220 07, Lund, Sweden
defense location
Chemical Centre, K:C
defense date
1996-09-20 10:30
external identifiers
  • Other:ISRN: LUTKDH/TKIT--96/1003-SE
ISBN
91-628-2146-6
language
English
LU publication?
yes
id
d9e25fc7-1156-4544-8b0f-7164a7fe8ef9 (old id 28612)
date added to LUP
2007-06-12 16:56:07
date last changed
2016-09-19 08:45:06
@phdthesis{d9e25fc7-1156-4544-8b0f-7164a7fe8ef9,
  abstract     = {In vitro generation of antibody fragments of desired specificity and affinity plays an important role since it permits the generation of reagents which may be valuable for diagnostic and therapeutic applications. Furthermore, the kinetic parameters for an antibody-antigen interaction, rather than the affinity, has shown to correlate with biological functions like virus neutralization. This points at the importance of being able to evaluate kinetic parameters. In this thesis we have evaluated the BIAcore biosensor for measuring affinity and kinetic constants and pointed at events where precautions need to be taken. The recently launched ORIGEN Analyser was, furthermore, evaluated for measuring affinity constants and a protocol for measuring dissociation rate constants was developed. The ability to select antibodies from phage displayed antibody libraries based on their kinetic parameters would be valuable. Two approaches in this direction were taken. The first was to use BIAcore biosensor for selection. By collecting elution fractions of an injected phage displayed antibody library, it could be shown that the time of dissociation is proportional to the dissociation rate constant. The second approach was to develop a modified protocol for SAP selection. SAP (selection and amplification of phage) links specific interaction to phage infection, by displaying the antibody fragment on a non-infectious phage and the antigen, fused to protein3, is added free in solution. Thus, only specific phages are allowed to infect and are amplified. We showed that addition of competing antigen to the interaction between antigen-protein3 fusion protein and a model phage displayed antibody library favoured low dissociation rate constants. Furthermore, a reduction in interaction time favoured high association rate constants. Thus, it was, for the first time, possible to select antibody fragments based on association rate. The last part of the thesis presents a new concept, which integrates phage and bacterial display in an attempt to physically link the genetic information of specifically interacting antibody-antigen molecules. This was performed by expressing peptide antigens on the surface of E. coli F pilus. The peptides were expressed as a fusion to pilin, the building block of pilus and encoded by the traA gene, which completely blocked protein3-mediated wild type phage infection. However, when a phage displayed scFv antibody was allowed to interact with pilus displayed peptide, we obtained a bacterial infection mediated by the specific antibody-antigen interaction. Thus, specific interaction on a protein-ligand level could be genetically rescued as a cellular linkage of the involved genes.This principle could have the potential to allow for screening of specific molecular interactions by crossing one phage and one bacterial library.},
  author       = {Malmborg Hager, Ann-Christin},
  isbn         = {91-628-2146-6},
  keyword      = {transplantation,affinity,antibody,kinetic,Immunologi,serologi,serology,Immunology,Bioteknik,Biotechnology,library,selection,surface display,phage display,association rate,dissociation rate},
  language     = {eng},
  pages        = {49},
  publisher    = {Ann-Christin Malmborg, Dept. of Immunotechnology, P.O.Box 7031, S-220 07, Lund, Sweden},
  school       = {Lund University},
  title        = {Molecular Recognition in Antibody Engineering. Studies on recombinant and phage displayed antibodies},
  year         = {1996},
}